Methods of diagnosing latent and active malignancies

ABSTRACT

Disclosed are procedures and methods for diagnosing latent and active cancers in a subject. The described methods include the use of sandwich ELISA assays containing antibodies specific for certain epitopes on the A-protein. This enables the assay to discriminate between the monomelic and homopolymeric forms of A-protein.

BACKGROUND OF THE INVENTION

This invention relates to methods for diagnosing both latent and activecancers. More specifically, this invention relates to methods fordiagnosing cancers by detecting the presence and relative amounts ofisoforms of A-protein in a subject.

A-protein is a cellular enzyme that was first isolated from vertebraterod photoreceptor cells by Schmidt et al., Invest. Ophthalmol. Vis.Sci., 24:244 (1983). A-protein is also known in the scientificliterature by the names G_(P), Cockcroft, Trends Biochem. Sci., 12:75-78(1987); recoverin Dizhoor et al., J. Biol. Chem., 267:16033-16036(1992); and CAR protein, Thirkill et al., Arch. Ophthalmol., 111:974-978(1993). A-protein has been characterized as a GTP-binding protein(g-protein), Schmidt et al., Invest. Ophthalmol. Vis. Sci., 28:94(1987), that regulates phosphinositide metabolism by activatingphospholipase C, Schmidt et al., Invest Ophthalmol. Vis. Sci., 29:123(1988).

A-protein exists in two forms; Schmidt et al., Invest. Ophthalmol. Vis.Sci., 30:172 (1989); and Dizhoor et al., J. Biol. Chem., 267:16033-16036(1992): as a monomer of 26,000 daltons which is soluble in the cytosol,and as a co-synthetically modified form to which a fatty acid isattached by the action of the enzyme N-myristoyl transferase (NMT;E.C.2.3.1.97). The modified form of A-protein tends to self-associate asstable pentameric homopolymers with an approximate molecular weight of130,000 daltons. These homoploymers are peripherally bound to the inneraspect of the cell membrane.

In its peripherally membrane-bound form, A-protein inactivated by agrowth-factor receptor imbedded in the plasmalemma subsequent toactivation of the receptor by a growth factor. The activation of thismetabolic cascade mechanism results in a sustained release of calciuminto the cytosol which ultimately stimulates the cell to divide. Thisgeneral scheme is referred to as signal transduction (see U.S. Pat. No.5,100,661).

In non-ocular tissues, A-protein transduces growth signals and isexpressed in mitotically active cells including malignant tissues.A-protein is expressed inside affected malignant cells and in the bloodstream. Fragments of the protein are also displayed on the surface ofmalignant cells. See Thirkill et al., Invest. Ophthalmol. Vis. Sci.,33:2768-2772 (1992).

U.S. Patent Publication No. 20070053893, published Mar. 8, 2007, toSchmidt, discloses a method for reducing immunological tolerance tomalignancy using formulations of myristoylCoA and N-myristoyltransferase to treat carcinomas displaying A-protein.

It will be readily appreciated that there exists a need for new andimproved methods for diagnosing both potential or latent cancers andactual malignancies which may be difficult to detect using conventionaldiagnostic tools.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the presence of thepentomeric homopolymer of A-protein as a method for diagnosing latentpre-cancer disease in a mammal.

The present invention also provides methods for detecting the presenceof the monomeric isoform of A-protein as a method for diagnosing anactive malignancy in a mammal.

According to one aspect of the invention, the presence of the A-proteinhomopolymer is detected from a sample of biological fluid obtained froma mammal, preferably a human. The sample is introduced into a sandwichELISA immunoassay which contains a capture antibody and a detectableantibody. The capture antibody and detectable antibody recognize thesame epitope on the A-protein, said epitope having the sequenceTDPKAYAQHV (SEQ ID NO:1). Excess unbound antibody is removed from theassay, and the amount of detectable antibody bound to the supportsurface is measured and compared to a standard for the assay. Anabnormally high level of the pentomeric homopolymer detected is a directmeasure of latent pre-cancerous disease in the mammal.

In a further aspect, the presence of the monomeric isoform of A-proteinis detected from a sample of biological fluid obtained from a mammal,preferably a human. The sample is introduced into a sandwich ELISAimmunoassay which contains a capture antibody and a detectable antibody.The capture antibody and detectable antibody recognize differentepitopes on the A-protein. In this aspect, the capture antibodyrecognizes an epitope unique to the monomeric, unmodified isoforms ofA-protein, while the detectable antibody recognizes the epitope havingthe sequence TDPKAYAQHV (SEQ ID NO:1). An abnormally high level of themonomeric form of A-protein detected compared to a standard is a directmeasure of active malignancy in a subject.

Preferably, the antibodies used in the immunoassays are eithermonoclonal antibodies, or antibody fragments, such as Fab fragments.Humanized and engineered or recombinant antibodies can also be used inthe immunoassay to advantage. The support surface can be a slide, plateor microtiter well. The individual immunoassay can also be part of aseries of immunoassays conducted on multiple human subjects usingrobotic or automated techniques well known in the art for large volumeassays.

Preferably, the detectable marker is a fluorescent dye or compound, orradioactive label, which can be readily detected and quantified usingknown techniques. The biological fluid can blood, serum, urine or lymphfluid.

In a still further aspect of the present invention, the antibodiesdescribed above are coupled to imaging agents using known techniques toallow clinicians to locate an active malignancy or the site of a latentmalignancy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of A-protein concentration for tissue samples obtainedfrom normal patients or patients in remission (controls), and patientssuffering from breast cancer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods for the detection of both latentand active cancers in a mammal. The types of active cancers which can betreated by the formulations described herein include, but are notlimited to, squamous cell carcinoma, small and large cell carcinoma ofthe lung, and breast, colon, cervical, and prostate carcinomas, as wellas primary or metastatic tumors. As used herein, the term “primarytumor” refers to tumor growth at a first site and not secondary togrowth elsewhere, while “metastatic tumor” refers to tumor growth at asite other than the original growth, caused by the migration ofmalignant cells from the first growth.

Unacylated or monomeric A-protein is expressed in the interior and onthe surface of malignant cells, such as small and large cell carcinomaof the lung and breast, prostate, colon, cervical and squamous cellcarcinomas. Acylation distinguishes the immunosuppressive (unacylated)form of A-protein from the immunogenic (acylated) form.

As used herein, the term “unacylated A-protein” refers to unmodifiedmonomers of A-protein having an apparent molecular weight onpolyacrylamide gels of 26,000 daltons.

The term “latent cancer” as used herein indicates that although thesubject does not currently have an active form of cancer, the cancer isessentially dormant and a subject has a high propensity for developingcancer at some time during their life expectancy. The dormant cancer canbe activated by any number of environmental or genetic triggers. Theterm “active cancer” indicates that the subject already has an activeform of cancer which has not been previously detected using conventionaltesting protocols.

The present invention utilizes a sandwich ELISA (Enzyme-LinkedImmunoSorbent Assay) to detect the presence of the monomeric orhomopolymeric forms of A-protein. The construction of such a sandwichimmunoassay is well known to those skilled in the art.

Briefly, antibodies to the monomeric and/or homopolymeric form ofA-protein can be obtained as described herein. One antibody isimmobilized on a solid support (the “capture antibody”). The support iscontacted with a sample of a biological fluid, and excess fluid isremoved from the assay. An unknown amount of antigen (an isoform ofA-protein) is immobilized on the solid support, and a second antibody(the “detection antibody”) is contacted with the support, forming acomplex with any antigen bound to the solid support. The detectionantibody can be covalently linked to a suitable marker, such as anenzyme, a photochemical compound, or a radioactive compound. The markeris detected using a detectable signal such as a fluorescent chemicalwhich emits fluorescent light when irradiated by a light source. Theamount of fluorescence detected is a direct measure of the amount ofantigen in the fluid sample.

The determination as to whether a particular cancer is latent, i.e.predisposed, or active depends on which particular isoform of A-proteinis detected (monomeric or homopolymeric), and the amount of A-proteinisoform so detected. The detection of the homopolymeric form in elevatedlevels as compared to a suitable reference standard is a positiveindication that a subject has a latent form of cancer. Alternatively,the detection of the monomeric isoform of the antigen in elevated levelsas compared to a reference standard is a positive indication that asubject has an active form of cancer.

The present invention can also be used to identify affected tissue invivo. This can be achieved by coupling the antibodies described above toimaging agents using known techniques. The imaging agents of theinvention are, in general, chemical entities which, when targeted to atumor or process, can be detected using appropriate imaging instruments.The antibody/imaging agent complex is then used by a clinician todetermine whether a mass is benign or malignant, and to locatemetastatic cancer sites in vivo.

In one embodiment, antibodies to the monomeric, unacylated isoform ofA-protein, and preferably antibodies to an epitope of A-protein that isunique and accessible only on the monomeric isoform of A-protein, arecoupled to imaging agents and used as a diagnostic tool to locate activemalignancies in a subject.

In another embodiment, antibodies to the homopolymeric, acylated isoformof A-protein, and preferably antibodies to the TDPKAYAQHV (SEQ ID NO:1)epitope of A-protein, are coupled to suitable imaging agents and used asa diagnostic tool to locate tissue within a subject having a latentpredisposition for developing a malignancy.

By “subject” is generally meant, in the context of this application, amammalian subject, and in particular, a human subject.

It is believed that antibodies against one isoform of A-protein arespecific to that form and do not recognize the other isoform. It has nowbeen discovered that the immunodominant epitope of A-protein thatcharacterizes that homopolymeric form has the amino acid sequenceTDPKAYAQHV (SEQ ID NO:1), and that the monomeric form has an epitopethat is not accessible on the polymeric isoform. Consequently, asandwich ELISA assay designed to detect the monomeric form of theprotein has antibodies directed to different epitopes of the protein: acapture antibody specific for the unique monomeric epitope, and adetection antibody specific for the TDPKAYAQHV(SEQ ID NO:1) epitope.Alternatively, a sandwich ELISA assay designed to detect thehomopolymeric isoform of the protein utilizes the same capture anddetection antibodies, both being specific to the epitope having thesequence TDPKAYAQHV(SEQ ID NO:1), thus rendering the assay specific forthe homopolymeric isoform.

In cancer cells, the regulatory pathway controlling cell division isgenerally absent, while this mechanism is present in normal maturecells. This results in the overproduction of A-protein and an upset ofthe normal equilibrium between the monomer and the polymer in cancercells, increasing the relative amount of monomer in the cytoplasm oftransformed cells relative to normal cells. It is believed that the lackof cellular differentiation seen in cancer cells causes the partial ortotal loss of the ability of the cell to modify A-protein, (see Olsen etal., J. Biol. Chem., 260:3784-3790 (1985)), i.e. the change fromimmunoreactive, modified A-protein to immunosuppressant, unmodifiedA-protein correlates strongly with the earliest stages of tumorformation.

Antibodies to both the monomeric and homopolymeric isoforms of A-proteincan be obtained using conventional techniques well known to thoseskilled in the art. Both monomeric and polymeric isoforms of A-proteincan be obtained, for instance, from biological sources such as bovineretinas as described in more detail in Schmidt et al., J. Biol. Chem.262:14333-14336 (1978).

Alternatively, isoforms of A-protein can be obtained by cloning a humangenomic or retinal library according to the methods of Dizhoor et al.,J. Biol. Chem., 267:16033-16036 (1992). Briefly, this entailsconstructing oligonucleotide probes that are complementary to portionsof the cDNA sequence of A-protein. See, for instance, Polans et al., J.Biol. Chem., 112:981-989 (1991). The library is expanded by polymerasechain reaction and expressed in a host vector such as E. coli which hasthe human DNA subcloned into its chromosomes. Bacteria are grown inculture dishes and the plaques are screened with the positive plaquesare selected and rescreened at least two more times. The gene product ofselected plaques is checked for the correct sequence, corresponding tothat of A-protein.

A-protein can also be cloned from a human genomic or retinal libraryaccording to the method of Ray et al., Proc. Nail. Acad. Sci.,89:5705-5709 (1992). Briefly, this procedure entails constructingoligonucleotide probes that are complementary to portions of the cDNAsequence of A-protein. The library is expanded by polymerase chainreaction and expressed in a host vector such as E. coli which has thehuman DNA subcloned into it's chromosomes. Bacteria are grown in culturedishes and the plaques are screened with the oligonucleotide probes.Positive plaques are selected and rescreened two more times. The geneproduct of selected plaques is checked for the correct sequence,corresponding to that of A-protein.

Example

A study is conducted using normal tissue specimens mixed with breastcancer tissue specimens. The normal tissue is obtained from normalpatients and patients in cancer remission (no evidence of disease), andis used as a control in this experiment. All tissue samples are testedin triplicate, and the results are returned to the clinical investigatorprior to breaking the code.

In the study of active malignancies, A-protein concentrations of morethan 10 ng/ml are considered positive for the assay. The results of thestudy are shown in FIG. 1, in which numerical values are recorded andgraphed. In FIG. 1, “Ned” indicates that there is no evidence ofdisease, while “Mets” indicates that the sample is diagnosed as havingmetastatic cancer.

As can be seen from FIG. 1, one specimen from the normal population isdiagnosed as a positive; indicating that the donor has an undiagnosedcancer. One specimen from the identified breast cancer patients sampled,one patient tested negative for the active disease, but positive for thelatent disease, demonstrating the higher level of information availableto the clinician, and the ability of the present diagnostic techniquesto discriminate between the metastatic recurrence of cancer from thenon-recurrent forms.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the appended claims. The patent and scientific literature referred toherein establishes the knowledge that is available to those with skillin the art. The issued U.S. patents, allowed applications, publishedforeign applications, and references cited herein are herebyincorporated by reference in their entirety.

1-29. (canceled) 30: A method for detecting the presence of themonomeric form of A-protein in a biological sample from a mammal, saidsample containing both the polymeric and monomeric forms of A-protein,said method comprising the steps of: contacting the sample with anA-protein capture antibody bound to a support surface, said captureantibody recognizing an epitope of A-protein present on the monomericform but not on the polymeric form of A-protein, removing excess samplefrom the support surface, contacting the support surface with anA-protein detectable antibody containing a detectable marker, saiddetectable antibody recognizing the epitope sequence of SEQ ID NO:1,removing unbound antibody from the assay, and determining the amount ofdetectable antibody bound to the support surface as a measure of theamount of the monomeric form of A-protein in the sample, and as ameasure of the active malignancy in the mammal. 31: The method of claim30 wherein all of the antibodies are monoclonal antibodies or antibodyfragments. 32: The method of claim 30 wherein the support surface is aslide, plate or microtiter well. 33: The method of claim 30 wherein thedetectable marker is a colorimetric enzyme, a fluorescent label or aradioactive label. 34: The method of claim 30 which is an immunoassayand wherein the sample is obtained from the bodily fluid. 35: The methodof claim 30 wherein an abnormally high level of the monomeric form ofA-protein detected compared to a standard is a measure of activemalignancy in the mammal. 36: The method of claim 30 wherein the mammalis a human subject. 37: The method of claim 30 wherein the fluid isselected from the group consisting of blood, serum, urine or lymphfluid.